Enterococcus faecalis is a significant agent in the pathogenesis of root canal infections, especially in post-endodontic treatment, with a prevalence of 24–77% in these infections ¹ . This bacterium is very difficult to eliminate because the pathogen can survive in poor nutrient conditions. It can adapt to acidic conditions, including living in the dentin tubule of a closed root canal with a smear layer. It can also express the dominant biofilm protein to maintain its attachment to host cells ² .

E. faecalis has been shown tolerate to acidic environments as well as to adapt to pH changes, which are the essential virulence factors in maintaining antibacterial balance ³ . Fisher reported that E. faecalis could survive in environments with high NaCl concentrations at extreme temperatures of 5–65°C with a pH of 4.5–10.0 ⁴ . Stuart et al. ¹ reported that E. faecalis are less sensitive, with a pH of 5.0 at 25°C after it has been incubated for 10 h. The author also found that it has an excellent growth capability at pH 8.5 and low adhesion at pH 7.1 in a medium coated with bovine serum albumin (BSA).

E. faecalis is resistant to medication materials such as calcium hydroxide ⁵ and chlorhexidine (CHX) ⁶ . The long-term use of both medication materials can lead to parachloroaniline (PAC), causing blockage of the dentinal tubules and eventually becoming toxic ⁷ . Fosfomycin may also interfere with acid tolerance systems and pH changes of E. faecalis in tooth root canals by inhibiting phosphoenolpyruvate synthetase ⁸ .

Indonesia, especially in Aceh has a tropical climate with a variety of plants that can be utilized in medical treatment, including lime ( Citrus aurantiifolia). Lime peel extract contains phenols, flavonoids, hydrogen peroxide, tannins, alkaloids, and saponins that have antibacterial, antioxidant, antifungal, analgesic, and anti-inflammatory properties ⁹ . Nwankwo ¹⁰ reported that lime extract helped to prevent Klebsiella pneumonia, Salmonella, and Escherichia coli. Here, the acid in lime extract influenced the bacterial development and cell metabolism. The present study evaluates the acid tolerance response and pH adaptation of E. faecalis when the bacterium grows as biofilm in the presence of lime extract with different concentration.

The experiment was performed in triplicate wells. This study showed that the presence of lime extracts decreased pH, but reduction of low pH did not have a significant effect on the ability of E. faecalis to adhere and form biofilm, compared to the control (fosfomycin). All results (adaptation to pH, acid tolerance assays and interaction activity are shown in Figure 1– Figure 3). Interestingly, the tolerance effect was not influenced by exposure time and the concentrations of lime peel extract set in this study ( Figure 3), and the correlation between time exposure and lime extract concentration was positive (r2 = 0.98).

E. faecalis did not express an ability to adapt to result in pH changes after interacting with fosfomycin (as a positive control) ( Figure 1), although it still expressed acid tolerance response ( Figure 2) and robust adhesion activity ( Figure 3).

In searching for pH adaptation of E. faecalis to ethanolic extract of lime peel, the initial pH of concentrated extract (100% v/v) without E. faecalis was 2.89. This is in agreement with those reported by Sitanggang et al. ¹⁹ that the water extract of lime has highly acidic pH ranges (1.7–3.1). As illustrated in Figure 1, this highly acidic pH only slightly changed under serial dilution and after the addition of E. faecalis suspension prior to incubation (0 hour).

This relatively stable pH of lime extract after serial dilution in water and after the addition of E. faecalis might be related to the presence of buffering compounds in the extract that is able to maintain pH. According to Bolhari et al. the juice of lime ( C. aurantiifolia) contains 88% water, 6–8% citric acid, 2% potassium citrate and calcium, 0.4–0.6% and other substances ²⁰ . The water extract of the fruit contains a number of bioactive compounds such as alkaloids, phenols, flavonoids, steroids, terpenoids, reducing sugar, saponins and cardiac glycosides ²¹ . Moreover, the peel of C. aurantiifolia contained 7 percent essential oil consisting of 46 compounds, most of which are terpenes ²² . The acidity is generated by citric acid and amino acids, while the essential oils contribute to maintaining its acidic pH ²³ . Citric acid is reported to play a crucial role as a natural material to maintain pH balance and possesses antibacterial activity ²⁴ .

After incubation there was a significant increase in the acidic pH of lime extract containing E. faecalis compared to lime extract alone (as a negative control) (p < 0.05). The increased pH occurred in all different concentration of lime extracts, from 6.25% (v/v) to 100% (v/v) (see Figure 1) indicates that E. faecalis can adapt to environments with an acidic pH (2.9–4.2) at a temperature of 37°C. The ability of E. faecalis to adapt to situations with a low pH and temperature has been reported by Morandi et al. ²⁵ in the experiment using pH adjusted to pH of 5.0 at 25°C within 10 hours.

Whilst better E. faecalis growth was observed in different lime extract concentrations with long exposure, increased acid tolerance response shown by the microbe as the concentration of the lime extract increased ( Figure 2) (p < 0.05). These phenomena probably relate to the ability of E. faecalis to produce a number of virulence factors in the extreme environment, such as the presence of potent antimicrobial agents or highly acidic condition ²⁶ . Among them is lipoteichoic acids that contribute to biofilm formation, a bacterial community-based resistant mechanism developed by certain bacteria to survive in extremely disadvantages environment ²⁷ .

Molecularly, the acid tolerance response of E. faecalis is influenced by the EfCitH gene, which encodes the citrate transporter protein on the surface of the bacterial cell membrane that acts to maintain the balance of the effects of citric acid generated from the environment ²⁸ . Sarantinopoulos found that enterococcal strains have metabolic potential against the citrate metabolism; this supports their acid tolerance response to environmental influences such as aroma and fermentation products ²⁹ . In this research, in the presence of fosfomycin with a pH of 7.2 ( Figure 1) E. faecalis could still slightly show pH tolerance. The acid tolerance response is related to the ability of certain E. faecalis strains to grow in environments with an alkaline pH (9.5–12) within 48–72 hours ¹² .

In general, E. faecalis showed progressively decreased adhesion ability in the presence of different concentrations of lime extract within 6 – 24 hours ( Figure 3). The adhesion started to increase after the microbe was exposure to different concentration of lime extract for 24 hours. The relatively high error bar (standard deviation values) obtained indicates such variation might be existed among the E. faecalis isolates toward the pH and concentration of lime extracts.

Varoni et al. ³⁰ reported that anti-adhesion activity between plant polyphenol-rich extract and Streptococcus mutans bacteria was at its maximum within 24 hours, while within 6, 7, and 8 hours, the activity was stable but not yet maximal. This is probably caused by the adaptation and tolerant mechanisms developed by E. faecalis against bioactive compounds presence in the extracts. Lime peel extract contains a number of metabolites (phenols, flavonoids, hydrogen peroxide, tannins, alkaloids, and saponins) have various therapeutic properties such as antibacterial, antioxidant, antifungal, analgesic, and anti-inflammatory ⁹ .

The mechanism utilized by bacteria to survive heat and low-pH of the environment operate in many different ways. The most successful means of surviving low-pH stress is the complete avoidance of extremely acidic environments. However, none more critical than the sensing of mild acidification to prevent the potentially lethal consequences of the inappropriate production of potentially antigenic proteins. Bacteria that are forewarned by mild acidification can prepare through the induction of a wide range of protective measures. It can alter the composition of the cell membrane, extrude protons, protect macromolecules, alter metabolic pathways, and generate alkaline ³¹ .

The lower adhesion activity observed in E. faecalis exposed to different concentrations of lime extract compared to that exposed to fosfomycin indicated better antibacterial activity of lime extract on the microbe than that of fosfomycin. Among active ingredients contained in the lime extract are flavonoids (polyethoxylated flavones and flavanones), coumarin, and terpenoids, all of which act as antibacterials ³² . Extract prepared from peel, fruit and leaves of lime show promising antibacterial activity against some microbes belong to both gram positive bacteria ( Staphylococcus aureus, S. epidermis and E. faecalis) and gram negative bacteria ( Klebsiella pneumonia and Proteus vulgaris) ³³ .

Enterococcus faecalis can adapt to environments with a pH of 2.9–4.3 generated by lime extracts. In addition E. faecalis also expressed a tolerance response to the acidic environment. The interaction activity of E. faecalis in different concentrations of lime extract become stable within 6–12 hours at a temperature of 37°C. Therefore, the lime extract can be used to inhibit the E. faecalis growth.